It is not always practical or desirable to transfuse a patient with donated blood. In these situations, use of a red blood cell substitute is desirable. Such a product would need to transport oxygen, just as red blood cells do.
When patients lose blood, it is usually necessary to replace the entire fluid volume lost. However, it is not usually necessary to replace all of the lost hemoglobin. The primary goal of hemoglobin replacement therapy is to transport oxygen from the lungs to peripheral tissues. Hemoglobin administration also increases and maintains plasma volume and decreases blood viscosity. While many volume expanding colloid and crystalloid solutions are now marketed, none can transport oxygen. The only current therapy with this capability is human blood transfusion.
In clinical practice, patients suffering acute loss of small to moderate amounts of blood require only volume resuscitation. More severe blood loss requires both volume replacement and replacement of oxygen carrying capacity. Only in situations such as massive blood loss, is it necessary to replace other blood components, such as platelets and dotting factors.
The following risks and limitations are currently associated with human blood transfusions:
1) Risk of infectious disease transmission (i.e., human immunodeficiency virus (HIV), non-A and non-B hepatitis, hepatitis B, Yersinia enterolitica, cytomegalovirus, Human T-cell Leukemia Virus 1). PA1 2) Immunologic risks (i.e., mild hemolytic or fatal transfusion reaction, immunosuppression, graft versus host reaction). PA1 3) Need for typing and cross-matching prior to administration. PA1 4) Availability of volunteer human donors. PA1 5) Limited stability (unfrozen shelf life 42 days or less).
Genetic engineering techniques have allowed the expression of heterologous proteins in a number of biological expression systems, for example, insect cell lines, transgenic cells, yeast systems and bacterial systems. Expression of hemoglobin in particular has recently been demonstrated in transgenic pigs (Logan, et al., WO 92/22646), yeast (De Angelo et al., WO 93/08831 and WO 91/16349; Hoffman et al., WO 90/13645), and the bacterial E. coli system (Hoffman et al., WO 90/13645). Although expression of hemoglobin in these heterologous systems can be achieved at high levels (i.e., in the range of 5-10% of total cellular protein), purification of the final product to the extreme level of purity required for pharmaceutical use of hemoglobin remains difficult. Removal of contaminating isoforms of hemoglobin is particularly difficult in that these isoforms often co-purify with the desirable form of hemoglobin.
Hemoglobin (Hb) is a tetrameric protein molecule composed of two alpha and two beta globin units. In fully functional, normal or native hemoglobin, a heme molecule is incorporated into each of the alpha and beta globins. Heme is a large organic molecule coordinated around an iron atom. A heme group that is lacking the iron atom is known as protoporphyrin IX (PIX). PIX can be incorporated into one or more of the .alpha. and .beta. subunits of hemoglobin, but the PIX-containing subunit lacks the ability to bind and release oxygen.
Alpha and beta globin subunits associate to form two stable alpha/beta dimers, which in turn loosely associate to form the hemoglobin tetramer. Human hemoglobin Ao (also known as naturally occurring or native hemoglobin) is a heterotetramer composed of two alpha globin subunits (.alpha..sub.1, .alpha..sub.2) and two beta globin subunits (.beta..sub.1, .beta..sub.2). There is no sequence difference between .alpha..sub.1 and .alpha..sub.2 or .beta..sub.1 and .beta..sub.2. In the unoxygenated ("deoxy", or "T" for "tense") state, the subunits form a tetrahedron. The .alpha..sub.1 .beta..sub.1 and .alpha..sub.2 .beta..sub.2 interfaces remain relatively fixed during oxygen binding, while there is considerable flux at the .alpha..sub.1 .beta..sub.2 and .alpha..sub.2 .beta..sub.1 interfaces. In the oxygenated ("oxy" or "R" or relaxed) state, the intersubunit distances are increased. The subunits are noncovalently associated by Van der Waals forces, hydrogen bonds and, for deoxy Hb, salt bridges.
Because the alpha and beta globin sequences of hemoglobin are known, and efficient expression criteria have been determined, it is possible that any suitable biological protein expression system can be utilized to produce large quantifies of recombinant hemoglobin. Indeed, hemoglobin has been expressed in a number of biological systems, including bacteria, yeast and transgenic mammals. However, expression of functional hemoglobin in any cell requires not only the expression of the alpha and beta globin protein segments but also incorporation of the heme group into each of the alpha and beta subunits. If hemoglobin contains a protoporphyrin IX molecule rather than a heme group in any of the four subunits, functionality is reduced. If all of the prosthetic groups are protoporphyrin IX rather than heme, then the hemoglobin cannot bind or release oxygen and is completely non-functional.
Hemoglobin has been purified from a number of sources, including outdated red blood cells from both human and other mammalian sources (see for example Estep, U.S. Pat. Nos. 4,861,867 and 4,831,012; Rausch et al., U.S. Pat. No. 5,084,558), yeast systems (see for example, De Angelo et al., WO 93/08831 and WO 91/16349; Hoffman et al., WO 90/13645), transgenic systems (see Logan, et al., WO 92/22646) and bacterial systems (see for example Hoffman et al., WO 90/13645). Purification of hemoglobin from all the above sources generally requires at least some lyric step to liberate the hemoglobin from the cellular matrix, a low resolution fractionation step to remove contaminating soluble and insoluble proteins, lipids, membranes, etc. (e.g. filtration, centrifugation, pH dependent precipitation and long term (&gt;1 hour) heating (see Estep U.S. Pat. No. 4,861,867), followed by some form of chromatographic final purification step known to those skilled in the art. The utilization of heat in the purification of hemoglobin from contaminating non-hemoglobin proteins is described in the Estep patent cited above. However, this fractionation of hemoglobin is useful only in the purification of already semi-pure hemoglobin solutions (99% by weight hemoglobin protein) that are in the deoxygenated (or T) state and that are derived from mammalian red blood cells heated for at least an hour, and can be considered a secondary purification step rather than an initial low resolution fractionation process. A dearly stated requirement of Estep is that the hemoglobin be in the deoxy state (hemoglobin which has no ligand, such as oxygen, bound to it) and that it be maintained in the deoxy or reduced state throughout the heating process by utilizing either chemical reductants or physical means (exposure to an inert gas).
When heterologous proteins are expressed in bacteria, heating of lysates of the bacterial cells, particularly E. coli lysates, is a common technique utilized in the purification of proteins derived from recombinant technology. However, heating of the material in solution after lysis of bacterial cells has generally been restricted to purification of known heat-stable proteins. This technique exploits the differences in thermal stability between most bacterial proteins and the heterologous protein. For example, in 1981 Tanaka and co-workers (Tanaka et al., (1981) Biochemistry 89: 677-682) expressed 3-isopropylmalate dehydrogenase from a thermophilic bacterium in E. coli, and purified this enzyme by heating the crude lysate for 10 minutes at 70.degree. C. They note that this was a simple and effective procedure for rapidly purifying protein, and further state that "the enzymes of extreme thermophiles are stable in conditions where most of the proteins of E. coli cells used as host are heat denatured and precipitated . . . these observations suggest that any thermophilic enzyme can be purified with relative ease by cloning the genes in question into E. coli."
The heat purification of protein was again used by Beguin and others in 1983 (Beguin et al., (1983) Biochimie 65:495-500) to purify another heat stable protein (endoglucanase B) by a heat treatment at 60.degree. C. for 15 minutes.
Tsukagoshi and co-workers in 1984 (Tsukagoshi et al., (1984) Mol. Gen. Genet. 193: 58-63) also purified a heat stable protein expressed in E. coli. However, they found that the thermal stability of the .alpha.-amylase that they were purifying was ligand dependent. The thermal stability in the absence of Ca.sup.++ was approximately 10.degree. C. lower than in the presence of Ca.sup.++ (see FIG. 5, page 61). As a result, these workers added Ca.sup.++ to the medium prior to heating to enhance stability of the enzyme and to recover greater activity. Moreover, this paper also demonstrates that the media conditions can be manipulated in order that the protein of interest is or becomes more thermostable than the contaminating E. coli proteins.
It is of note that these systems require both (a) heating of the lysate solutions for at least 10 minutes, and (b) a significant difference between the thermal stability of most of the contaminating proteins and the protein of interest.
Charm, U.S. Pat. No. 4,975,246, discloses a method for rapid heating (usually one second or less) of heat-sensitive materials using microwave energy in order to achieve pasteurization or sterilization of the material. There is no mention of using the method to achieve selective removal of contaminating materials.
Therefore, the present invention has the unexpected and heretofore unobserved characteristic of separating desired proteins from a protein matrix using rapid heating when the desired protein has similar thermal stability to some of the proteins in the protein matrix.
It is important to note that hemoglobin containing protoporphyrin IX rather than heme had not been obtained prior to this invention other than hemoglobin which has been chemically treated or modified specifically to result in protoporphyrin IX-containing hemoglobin. We have discovered that hemoglobin solutions, particularly those hemoglobin solutions resulting from recombinant production of hemoglobin, do contain significant levels of contaminating protoporphyrin IX-containing hemoglobin. Moreover, no mechanism for removal of such contaminating poorly functional hemoglobin has been available until now. Surprisingly, we have discovered that hemoglobin containing protoporphyrin IX rather than heme can be separated from fully functional hemoglobin by subjecting mixtures of said hemoglobins to sufficient heat for a sufficient period of time.